Mechanistic Investigation of Lgals3+ Macrophages in Fracture Callus Fibrosis and Dysfunctional Repair

SUMMARY Fibrosis, characterized by excessive accumulation of extracellular matrix proteins, is a key feature of chronic inflammatory conditions that severely impairs organ function. Unlike most tissues, bone possesses a unique capacity for scar-free healing. However, complications such as fracture nonunion or delayed union can result in fibrotic tissue formation, leading to substantial disability. While fibrosis is typically associated with critical-sized bone defects, mechanical instability at the fracture site can also trigger fibrosis by sustaining chronic inflammation. The precise mechanisms underlying strain-induced fibrosis and compromised bone healing remain poorly understood. This proposal aims to uncover the biological processes that drive fibrosis in high-strain fracture environments and to develop targeted therapies for enhancing bone repair. Our preliminary studies using a murine fracture model with custom intramedullary nails to control mechanical strain show that strains above 15% induce fibrosis and poor healing, while strains below 5% support robust bone formation. Spatial transcriptomics revealed that high-strain conditions upregulate the pro-fibrotic marker Lgals3 and increase macrophage-fibroblast interactions. We hypothesize that fibrosis and dysfunctional bone repair in high-strain fracture environments are mediated by Lgals3-expressing macrophages through the secretion of galectin-3 (Gal3). To test this hypothesis, we will pursue two specific aims. In Aim 1, we will validate the role of galectin-3 in fracture callus fibrosis and bone repair using Gal3-deficient (Lgals3fl/fl; Lyz2-IRES-CreERT2) mice. We will evaluate fracture callus development, functional healing outcomes, and molecular indicators of fibrosis, comparing these findings with wild-type controls. In Aim 2, we will evaluate the therapeutic potential of Selvigaltin, a potent Gal3 inhibitor, to mitigate callus fibrosis and enhance bone healing. We will administer Selvigaltin at various stages post-fracture and assess its effects on fibrosis resolution and healing quality relative to saline-treated controls. This research will provide critical insights into the mechanisms of pathological fibrosis and dysregulated tissue repair in mechanically unstable environments. The findings will inform new therapeutic strategies to improve outcomes for patients with inflammatory bone disorders, advancing the field of regenerative medicine.